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Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 knockout
Philip H Iffland1, Allan E Barnes1, Marianna Baybis1
1Department of Neurology, University of Maryland School of Medicine, Baltimore, MD, United States of America.
Abstract:
TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein 1 (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been comprehensively investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR-associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.
Insights
Amino acid levels differentially affect mTOR signaling in neurons with Tuberous Sclerosis Complex (TSC) or epilepsy-associated gene mutations. This impacts potential treatments for mTOR-related neurological disorders.
Area of Science:
- Molecular Neuroscience
- Cellular Signaling
- Genetics and Disease
Background:
- Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, leading to mechanistic target of rapamycin (mTOR) hyperactivation.
- Amino acid (AA) levels are known to modulate mTOR signaling in non-neural cells, but their role in neurons is less understood.
- DEPDC5 mutations are linked to epilepsy and also affect mTOR regulation.
Purpose of the Study:
- To investigate how amino acid (AA) levels impact mTOR signaling and downstream targets (S6 and 4E-BP1 phosphorylation) in neurons.
- To compare the effects of AA levels and rapamycin on mTOR signaling in Tsc2-deficient versus Depdc5-deficient neuronal models.
- To elucidate the differential mechanisms underlying mTOR regulation by AAs in distinct genetic epilepsy models.
Main Methods:
- CRISPR-edited Neuro2a (N2a) cells and differentiated neurons lacking Tsc2 or Depdc5 were utilized.
- Analysis of S6 and 4E-BP1 phosphorylation under varying amino acid conditions (amino acid-free media).
- Utilized a CFP/YFP FRET-biosensor for real-time 4E-BP1 phosphorylation assays and confocal imaging for mTOR localization.
Main Results:
- Tsc2 knockout (KO) neurons showed reduced S6/4E-BP1 phosphorylation and cell size in amino acid-free (AAF) media, unlike Depdc5 KO neurons.
- AAF conditions promoted mTOR lysosomal displacement in Tsc2 KO cells but not Depdc5 KO cells, indicating differential mTOR localization.
- Rapamycin inhibited S6 phosphorylation but not 4E-BP1 phosphorylation in either KO model, suggesting distinct pathway modulation.
Conclusions:
- Amino acid availability differentially modulates mTOR signaling and lysosomal localization in Tsc2 KO versus Depdc5 KO neurons.
- Neuronal mTOR signaling in mTOR-associated epilepsies exhibits distinct responses to amino acid levels and mTOR inhibitors.
- Findings highlight the potential for targeted therapeutic strategies based on specific genetic underpinnings and nutrient availability.
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